Embryonic stem cells, referred to as ES cells, are derived from the inner cell mass (ICM) of fertilized eggs in blastocyst phase, and can be cultured and maintained in vitro while being kept in an undifferentiated state. ES cells are pluripotent, possessing the capability of developing into any organ or tissue type or, at least potentially, into a complete embryo. For example, ES cells can differentiate and give rise to a succession of mature functional cells. Differentiation has been shown in tissue culture and in vivo.
An important application of human ES cells is their use in cell therapy: the treatment of symptoms, diseases, conditions, and disabilities with ES cell derived replacement cells and tissues. Many diseases and disorders result from disruption of cellular function or destruction of tissues of the body. A wide spectrum of diseases may be treated based upon both the possession of a population of cells having multi-lineage potential and an understanding of the mechanisms that regulate embryonic cell development. Pluripotent stem cells that are stimulated in vitro to develop into specialized cells offer the possibility of a renewable source of replacement cells and tissue to treat numerous diseases, conditions, and disabilities. Some of these diseases, conditions, and disabilities include but are not limited to Parkinson's and Alzheimer's diseases and other neurodegenerative disorders, spinal cord injuries, stroke, macular degeneration, burns, liver failure, heart disease, diabetes, Duchenne's muscular dystrophy, osteogenesis imperfecta, osteoarthritis, rheumatoid arthritis, anemia, leukemia, breast cancer and other solid tumors, and AIDS.
ES cells have been derived from mouse (Evans and Kaufman, Nature 292:154-156, 1981; Martin, PNAS USA 78:7634-7639, 1981), hamster (Doetschmann et al., Dev. Biol., 127:224-227, 1999), sheep (Handyside et al., Roux's Arch. Dev. Biol., 198:48-55, 1987; Notarianni et al., J. Reprod. Fertil., 43:255-260, 1991; Piedrahita et al., Theriogenology, 34:879-901, 1990), cow (Evans et al., Theriogenology, 33:125-128, 1990), rabbit (Giles et al., Mol. Reprod. Dev., 36:130-138, 1993), mink (Sukoyan et al., Mol. Reprod. Devl, 36:148-158, 1993) and pig (Piedrahita et al., Theriogenology, 29:286, 1988; Evans et al., supra, 1990; Notarianni et al., J. Reprod. Fertil., Suppl. 41:51-56, 1990). Recently, the derivation of human ES cells has been reported (Thomson et al., Science, 282:1145-1147, 1998; Shamblott, et al, Proc. Natl. Acad. Sci. USA, 95:13726-13731, 1998; Reubinoff et al., Nature Biotechnology 18:399-404, 2000 (published erratum Nature Biotechnology 18:559, 2000)).
Human ES cells have been isolated from two different tissue sources, however, the characteristics of the derived ES and embryonic germ (EG) cells are very similar (reviewed in Pera et al., J. Cell Science, 113:5-10, 2000). Thomson et al. isolated ES cells from the ICM of surplus human blastocysts that had been donated from fertility clinics (Thomson et al., supra, 1998), while Shamblott et al. isolated stem cells from the gonadal tissues of terminated pregnancies (Shamblott et al., supra, 1998). In neither case were the blastocysts or embryos created for the purpose of research.
The ES cell isolated by Thomson et al., and the embryonic germ (EG) cell derived by Shamblott et al. are reported to share certain characteristics: the cells originate from a pluripotent cell population; they maintain a normal karyotype in vitro; they are immortal and can be propagated indefinitely in the embryonic state; and are capable of spontaneous differentiation into somatic cells representative of all three embryonic germ layers in teratomas or in vitro (reviewed in Pera et al., supra, 2000).
The culture conditions for the human ES and EG cells differ from the culture conditions for the mouse ES cell. Mouse ES cells are typically derived using fibroblast feeder layers. The fibroblast feeder layers typically are either STO fibroblasts, a transformed cell line, or more often, the mouse ES cell is co-cultured with a primary culture of mouse embryonic fibroblasts (MEFs). These cultures are typically supplemented with leukemia inhibitory factor (LIF). The mouse ES culture medium may alternatively be supplemented with other growth factors that prevent differentiation. Examples of such growth factors are OSM, CNTF, IL-6 in combination with soluble IL-6 R, or other cytokines that signal through the gp130 pathway.
Mouse ES cells remain undifferentiated indefinitely in the presence of an embryonic fibroblast feeder layer. Similarly, it is reported that a feeder layer consisting of mitotically inactivated MEFs or other fibroblasts is required for human ES cells to remain in an undifferentiated state (see e.g., U.S. Pat. No. 6,200,806; Amit et al., Developmental Biology 227:271-78, 2000; Odorico et al., Stem Cells 19:193-204, 2001). However, while mouse ES cells will also remain undifferentiated in the absence of an embryonic fibroblast feeder layer so long as the medium is supplemented with LIF (Smith et al., Nature 336:688-690, 1988; Williams et al., Nature 336:684-687, 1988), human ES cells differentiate or die in the absence of a fibroblast feeder layer, even when the medium is supplemented with LIF (Thomson et al., 1998 supra; Reubinoff et al., 2000 supra).
The exact role of the MEFs in establishment and maintenance of a ES cell culture is not known. Possible roles for the MEFs include prevention of differentiation or death, or induction of proliferation, by one or some of a number of mechanisms, including, but not limited to the production of cytokines such as LIF, the provision of extracellular matrix components that provide attachment sites for the ES cells, the provision of receptor-style interactions that provide survival signals for the ES cells, the presentation of cytokines to the ES cells, the adsorption of environmental toxins such as heavy metals, or the secretion of growth factors necessary to support the ES cell.
While fibroblast feeder layers are critical to the survival and non-differentiation of the human ES cell, mouse embryonic fibroblast feeder cells are labor-intensive to derive, and can vary between lots (Amit et al., supra, 2000). The development and use of non-fibroblast feeder cell layers that are not labor-intensive to establish, and that offer greater consistency than embryonic fibroblast cells would be an advantage to the field. Moreover, the potential applications for the human ES cell are limited when the ES cell is cultured in the presence of non-human feeder cell layers. Ideally, a human ES cell could be cultured with human feeder cell layers, or could be cultured in the presence medium conditioned by human cells.
There is no evidence in the prior art showing the long-term isolation and/or maintenance of human pluripotent ES cells on non-fibroblast feeder cells. Others have attempted to isolate human ES cells on non-fibroblast feeder cells, but have not succeeded in maintaining the human ES cells in a pluripotent state for long or indefinite periods of time. Bongso et al. cultured human blastocysts on oviduct epithelial cells in the presence of human LIF (Bongso et al., Human Reproduction 9:2100-2117, 1994). Bongso et al. then separated the ICMs from the trophoblast and feeder cells, and replated the ICM-derived cells in the absence of a feeder layer. This method supported the growth of ICM-derived cells for two subcultures, or at least 18 days, without differentiation; however, the cells subsequently differentiated into fibroblasts or died.
Similarly, there is no evidence in the prior art showing the long-term isolation and or maintenance of human pluripotent ES cells in the presence of conditioned media from human cell types. Although the co-culture of human ES cells with conditioned media from mouse embryonic fibroblasts has been reported (Xu et al., Keystone Symposia Abstract Book, Pluripotent Stem Cells: Biology and Applications, February 2001, A. 133), conditioned medium from human cell cultures has not been reported to maintain human ES cells in a pluripotent state.
Granulosa cells are the cells that support and nourish the oocyte in the ovary. Granulosa cells are thought to arise from a population of stem cells (Rodgers et al., Mol Cell Endocrinol 22;171(1-2):41-8, 2001; Lavranos et al., Biology of Reproduction 61, 358-366, 1999; Rodgers et al., J Reprod Fertil Suppl 54:343-52, 1999). Initially, a primordial follicle consists of an oocyte surrounded by a single layer of flattened epithelial pregranulosa cells. As the follicle grows, the granulosa cells proliferate radially, reaching a total of tens of thousands of cells in the preovulatory state. Granulosa cells cease dividing at ovulation, and after ovulation, granulosa cells differentiate into the luteal cells of the developing corpus luteum in the ovary. See also generally, Weiss, et al., Eur J Endocrinol. 144(6):677-85, June 2001; Stevenson, Indian J Exp Biol. 2000 December;38(12):1183-91; Hosokawa et al., Endocrinol; 138(11):4679-4687, 1998; Hosokawa et al., Endocrinology 138(11):4688-4700, 1998; Byong-Lyul et al., Mol and Cell. Endocrinology 120:169-176, 1996.
Researchers have attempted to use pig granulosa cells as feeder cell to support the isolation and/or maintenance of pig and cow ES cells (Vasil'eva and Vasil'ev, 1995 Russian J. Dev. Biol., 26:167-72, Translated from Ontogenez, 26:206-12, 1995; Vasil'ev and Vasil'eva, 1995 Russian J. Dev. Biol., 26:163-66, Translated from Ontogenez, 26:201-205, 1995). Pig embryos did not attach to pig granulosa cells, and while pig embryonic cells did attach to pig granulosa cells, the cultured embryonic cells produced trophoblast-like cells and not ES-like cells (Vasil'ev and Vasil'eva, 1995 supra). Cow embryos did attach to pig granulosa cells, and formed ES-like cells that could be maintained in culture on granulosa cell feeder layers for three transfers without differentiating (Vasil'eva and Vasil'ev, 1995 supra). Thus the culture conditions which were successful with one large domestic animal were not successful for another domestic animal. The authors acknowledge that the techniques useful for the isolation of ES cells from large domestic animals will differ from those useful for the isolation of ES cells from mice. It is therefore not predictable that a technique successful for the isolation and short-term maintenance of ES-like cells from cows will be useful for the isolation and/or maintenance of human pluripotent stem cells.
For the treatment of many human diseases by cell therapy, it may be necessary to direct the differentiation of human ES cells in culture, prior to transplanting the ES cells into the subject. In vitro differentiation may be directed by the addition of supplemental growth factors to the culture medium.
Various soluble factors have been used to induce differentiation of mouse ES cells down specific lineages: IL-3 directs cells to become macrophages, mast cells or neutrophils (Wiles, M. V., and Keller, G., Development 111:259-267, 1991); IL-6 directs cells to the erythroid lineage (Biesecker, L. G. and Emerson, S. G., Exp. Hematol., 21:774-778, 1993); retinoic acid induces neuron formation (Slager et al., Dev. Genet. 14:212-224, 1993; Bain et al., Dev. Biol. 168:342-357, 1995); and transforming growth factor (TGF)-β1 induces myogenesis (Slager et al., supra, 1993; Rohwedel et al., Dev. Biol. 164:87-101, 1994). Most of these studies were performed on ES cells that had been induced to form embryoid bodies in culture (Slager et al., supra, 1993; Bain et al., Supra, 1995; Rohwedel et al., supra, 1994). While the use of the soluble factors induced differentiation of different cell lineages, the factors did not induce differentiation of only one cell type; instead, the factors changed the proportion of the different cell types in the cultures.
The most comprehensive analysis of human ES cells examined the effects of eight growth factors on the differentiation of cells grown first as embryoid bodies and then disaggregated (Schuldiner et al., 2000; PNAS USA 97:11307-11312). Schuldiner et al. applied basic fibroblast growth factor (bFGF), TGF-β1, activin-A, bone morphogenetic protein 4 (BMP-4), hepatocyte growth factor (HGF), epidermal growth factor (EGF), β nerve growth factor (βNGF), and retinoic acid to the cells, and determined the effects on cell-specific gene expression and cell morphology. TGF-β1 and activin-A induced differentiation of muscle cells; retinoic acid, bFGF, BMP-4, and EGF induced differentiation of ectodermal and mesodermal cells; while NGF and HGF allowed differentiation of cells from all three germ layer lineages. However, none of the growth factors tested directed the differentiation of a uniform and singular cell type.
Finally, Reubinoff et al. were able to isolate human neuronal-lineage cells in a relatively pure form from a human ES culture (Reubinoff et al., 2000 supra). The differentiation of neuronal-lineage cells occurred spontaneously when the human ES cell was cultured on mouse embryonic fibroblasts. Reubinoff et al. isolated the areas of differentiated cells and re-plated the cells in serum free medium. The cells formed spheres, which were again re-plated and allowed to attach to an adhesive substrate. Although this procedure provided a relatively pure population, these cells cannot be used for the treatment of humans since they were cultured on mouse feeder cells. Additionally, the differentiation was not directed towards a specific lineage. There is a need, therefore, to develop methods for the directed differentiation of human ES cells that are not cultured with mouse feeder cells. These methods may involve the addition of a supplemental growth factor to the culture medium.
There is a need, therefore, to establish culture conditions, such as human feeder cells, or conditioned medium, that allow for greater reproducibility and consistency among cultures, and that allow for the use of the human ES cells in cell therapies. There is also a need to establish methods for selectively differentiating human ES cells into precursors and into the desired and uniform cell lineages, such as the neuronal cell lineage. Large, purified populations of selectively differentiated ES cells will provide a potentially limitless source of cells for cellular therapy treatments and further drug discovery. Selectively differentiated, and reversibly differentiated, ES cells can be used for cell therapy, and transplanted into subjects to treat a number of different conditions and diseases.